Aberrant gene expression of superoxide dismutases in Chlamydia trachomatis-infected recurrent spontaneous aborters

Study aimed to characterize the expression of antioxidant genes SOD1 and SOD2 in Chlamydia trachomatis-induced recurrent spontaneous aborters and further determine their role by in silico analysis. First void urine was collected from 130 non-pregnant women with history of recurrent spontaneous abortion (RSA) (Group I) and 130 non-pregnant women (Group II; control) attending Obstetrics and Gynecology Department, SJH, New Delhi, India. C. trachomatis detection was performed by conventional PCR in urine. Gene expression of SOD1 and SOD2 was performed by quantitative real-time PCR. Further, its interacting partners were studied by in silico analysis. 22 patients were positive for C. trachomatis in Group I. Significant upregulation was observed for SOD2 gene in C. trachomatis-infected RSA patients while SOD1 was found to be downregulated. Increased concentration of oxidative stress biomarkers 8-hydroxyguanosine and 8-isoprostane was found in C. trachomatis-infected RSA patients. Protein–protein interaction (PPI) of SOD proteins and its interacting partners viz.; CCS, GPX1, GPX2, GPX3, GPX4, GPX5, GPX7, GPX8, CAT, PRDX1, TXN, SIRT3, FOXO3, and AKT1 were found to be involved in MAPK, p53 and foxo signaling pathways. Molecular pathways involved in association with SODs indicate reactive oxygen species (ROS) detoxification, apoptotic pathways and cell cycle regulation. Overall data revealed alleviated levels of SOD2 gene and decreased expression of SOD1 gene in response to C. trachomatis-infection leading to production of oxidative stress and RSA.

performed by the SYBR green-based chemistry to quantitate the C. trachomatis copies in urine of groups I-II patients. Standard curve was plotted by preparing serial dilutions using the known concentrations of C. trachomatis-positive control. A total of 2000-11,000 copies/ml were detected in the urine of C. trachomatispositive RSA (Fig. 1). 08, 04, 06, 02, 02 patients had a copy number of 2000-11,000, 4000-5000, 6000-7000, 8000-9000, 10,000-11,000/ml respectively.

Concentration of urine 8-OHdG and 8-isoprostane in recurrent spontaneous aborters. Mean
urine 8-OHdG level was estimated in controls and C. trachomatis-positive as well as uninfected RSA and it was found that the 8-OHdG concentration was significantly high (99.65 ng/ml) in the C. trachomatis positive RSA, as compared with both uninfected RSA (50.54 ng/ml) and the control group (31.97 ng/ml; Mann-Whitney test, 'p' < 0.05). Mean urine 8-isoprostane concentration was significantly high (808.28 pg/ml) in the C. trachomatis-positive RSA, as compared with both uninfected RSA (501.39 pg/ml) and control group (208.02 pg/ml; Mann-Whitney test, 'p' < 0.05) (Fig. 2).

Quantitative expression of SOD1 and SOD2 genes.
To determine whether C. trachomatis affects the expression of SODs at the transcript level in RSA patients, q-PCR was performed and the expression of Group Ia was compared with Group II. Constitutively expressed gene GAPDH was used as the endogenous control. Analysis revealed significant downregulated mRNA expression with a relative fold change of 0.52 for SOD1 in Group Ia (n = 22) as compared to Group II (n = 130) while fold change of 1.4 was observed in Group Ib (n = 108) as compared to Group II (n = 130) (Mann-Whitney non-parametric test; p < 0.05). In case of SOD2, analysis revealed significant upregulated mRNA expression with a fold change of 1.4 in Group Ia (n = 22) versus Group II (n = 130) whereas a fold change of 0.69 was found in Group Ib (n = 208) versus Group II (n = 130) (Mann-Whitney test-non parametric test; p < 0.05). (Fig. 3).
Correlation between genes SOD1 and SOD2 and gestational age (GA). SOD1 and SOD2 gene expression was correlated with GA. Positive significant correlation was observed between SOD2 and GA (r = 0.449, p = 0.038) while a negative correlation was observed between SOD1 and GA (r = − 0.462, p = 0.047) (Fig. 4).
Correlation between C. trachomatis load and SODs. The expression of SOD1 and SOD2 were correlated with the chlamydial load in the urine of infected RSA. A statistically significant positive correlation was observed between SOD2 and C. trachomatis copy load (r = 0.468; 'p' = 0.027). Chlamydial load and SOD1 gene was found to be negatively correlated in C. trachomatis-positive RSA (r = − 0.45, 'p' = 0.035) indicating that a greater C. trachomatis copy load leads to decreased expression of SOD1 gene in RSA patients (Fig. 5).
Dysregulated genes were integrated on Cytoscape software for biological function assessment and GO/ KEGG pathway enrichment. MCODE analysis of DEGs on Cytoscape revealed two clusters of network showing interaction with SOD1 and SOD2 genes (Fig. 7a, b). Molecular function, biological process and cellular component domains were covered under GO. In case of SOD1 gene and its interacting partners, regulation of  www.nature.com/scientificreports/ signal transduction and system processes was the most significant biological process (Table 2a). Dysregulated genes in RSA were a part of the intracellular cell component (Table 2b). Molecular function found to be most significant was protein/ enzyme binding (Table 2c). In case of SOD2 gene, cell proliferation, transcription and homeostasis were the significant biological processes for the dysregulated genes in RSA (Table 3a), while cellular component was intracellular (Table 3b) and protein binding was the significant molecular process (Table 3c). The results revealed that DEGs were markedly involved in MAPK, p53 signaling pathway, cell cycle pathway and chemokine signaling pathways (Tables 2d, 3d).

Discussion
RSA has been a challenging and a complex condition with idiopathic etiology. The etiology is unexplained and poorly understood in most of the cases thus making it difficult for physicians to treat. Homeostatic balance of antioxidants and oxidants is imperative towards maintaining normal cellular physiological functions. ROS are important for several physiological and cell signaling functions but also play a major role in pathology of reproductive processes. Their overproduction leads to cellular damage and can contribute to pathogenesis of diseases by impeding normal physiological activities of the cell. Previous studies have suggested that release in excessive oxygen radicals can lead to pregnancy complications such as pre-eclampsia, spontaneous abortion and also reproductive associated diseases such as polycystic ovarian syndrome, endometriosis [29][30][31] . SODs require the presence of oxygen and metal ions cofactors for their function. Present in subcellular localization; Cu-Zn SOD in the cytoplasm and extracellularly and MnSOD in the mitochondria 32 . Unsuccessful maintenance of pregnancy due to C. trachomatis infection places a major burden on women. The pathogen evades the upper genital tract causing scarring and occlusion of fallopian tube, pelvic inflammatory diseases and in extreme cases infertility. The host pathogen interaction involves an array of participating signaling molecules like cytokines and ROS. C. trachomatis induces the release of ROS causing series of events leading to cell damage which eventually progresses to chronic infection. Few studies have been conducted in C. trachomatisassociated OS in reproductive pathology. A study suggested C. trachomatis infection as a predominant factor in the pathogenesis of abortion as it increases production of ROS and thereby OS leading to unfavorable pregnancy outcomes 33 . A similar study reported lower antioxidant capacity in patients with tubal infertility C. trachomatispositive women compared to fertile C. trachomatis-negative control 34 .
In the present study, mRNA expression of SOD1 and SOD2 genes was determined in C. trachomatis-infected RSA and control patients in urine by q-RT-PCR. Our data confirmed significant upregulation of SOD2 gene expression in Group Ia when compared to Group II ('p' > 0.05) whereas decreased expression was observed in    A significant correlation was observed between SOD2 gene and GA in C. trachomatis-positive RSA patients while no significant correlation was observed between SOD1 gene and GA in C. trachomatis-positive RSA patients. Chlamydial load showed positive correlation with SOD2 while a negative correlation was observed with SOD1 gene suggesting that greater chlamydial load leads to decreased expression of SOD1 gene. Elevated levels of 8-OHdG and 8-isoprostane were observed in Group Ia when compared to controls (p < 0.05) indicating that these biomarkers might be involved in the underlying pathological mechanism adopted by C. trachomatis in inducing RSA.
Several studies have implicated the association of the above mentioned interacting partners in regulation of OS in RSA and C. trachomatis infection suggesting their role together with SOD genes. For instance, PRDX2, one of the interacting partners of SOD genes was reported to have significantly lower expression in trophoblast cells of RSA patients. Subsequent to knockdown of PRDX2 gene the cellular ROS levels increased which led to proliferation and apoptosis 35 . In another study PRDX3 and PRDX4 have been shown to play pivotal role in implantation and normal placentation through their antioxidant activity. The family of peroxiredoxins plays important roles in neutralizing OS. Peroxiredoxins also interact with thioredoxins by maintaining its reduction status and further downregulating pro-apoptotic pathways 36 . FOXO proteins (another interacting partner of SOD genes) are key regulators in cell cycle progression, cell differentiation, DNA repair, apoptosis and cell differentiation 37 and one of the most activated pathways during chlamydial infection 38 . In a study it has been demonstrated that decidualizing cells are resistant to oxidative cell death due to function of antioxidant genes particularly SOD2, thioredoxin, peroxiredoxin and if exposed to OS, it induces FOXO expression alleviating ROS mediated apoptosis 39 . Glutathione along with GPX plays a key role in determining the progression of C. trachomatis infection and its developmental cycle implying susceptibility to ROS 40 . These findings suggest that SOD genes with its interacting partners might be playing critical roles in pathology of C. trachomatis infection during implantation and pregnancy.
GO/KEGG analysis revealed that the DEGs were involved in biological and molecular processes such as protein binding, intracellular transduction signaling process, cell proliferation and transcription regulation. MAPK, cell cycle, chemokine and apoptotic pathways were involved for these DEGs found to be interacting with SOD1 and SOD2 genes. Genes enriched in the MAPK pathway were FGF7, IGF1, and KRAS which are known to be involved in RSA. These genes have a major role in migration, proliferation and invasion of cells www.nature.com/scientificreports/ during implantation. Similarly, the enriched genes of the p53 pathway were MDM4, CDK1 and IGF1 which have diverse functions in cell cycle progression and apoptosis. Therefore, dysregulation of these molecular pathways can result in pathogenesis of RSA. Higher levels of p53 were detected in placental villi resulting in apoptosis by mediating trophoblast infiltration and eventually spontaneous abortion 41,42 . These genes also play important role during C. trachomatis infection. C. trachomatis is highly dependent on MDM2-p53 interaction. Inhibition of the p53-MDM2 interaction disrupts intracellular development and interferes with the pathogen's anti-apoptotic effect on host cells 43 . C. trachomatis manipulates the eukaryotic cell cycle by destabilizing CDK1 proteins 44 . In another study, it was shown that FGF7 regulates proliferation of endometrial cells via the MAPK pathway 45 .
C. trachomatis has also been known to induce its infection by MAPK/ERK pathways by stimulating FGF and enhancing its infection and spread leading to apoptosis of host cells 46,47 . A finding suggests that the effect of C. trachomatis infection on trophoblast involves the chemokine signaling pathways leading to expression of innate immune receptors by the trophoblast and virulence factors secreted into the trophoblast by the bacteria 48 . This type of cross talk as in the case of infection induced inflammation could be responsible for C. trachomatisinduced spontaneous abortion. All these pathways finally diverge to major cellular processes such as cell cycle, apoptosis, DNA repair and ROS detoxification involving the enriched genes found in our study. It can be further

Methods
Ethical approval, enrolment of patients and collection of clinical sample. Ethical approval for the study was obtained from the Institutional Ethics Committee, VMMC and Safdarjung Hospital, New Delhi, India (IEC/VMMC/SJH/Project/2019/08/42). Thereafter, the present case-control study was undertaken in 130 nonpregnant women attending Department of Obstetrics and Gynecology, Safdarjung Hospital, New Delhi, India having a history of RSA in the first trimester (Group I) while 130 age-matched asymptomatic non-pregnant women having history of two successful deliveries served as controls (Group II). Written consent was obtained from each patient prior to collection of samples. Recurrent aborters with recent antibiotic therapy, chromosomal abnormalities, autoimmune diseases such as diabetes, metabolic disorders like thyroid and anatomical factors like endometriosis and uterine anomalies were excluded from the study. Patients with previous history of genitourinary tract infection, HIV-positivity, VDRL-positivity, TORCH (Toxoplasma gondii, Rubella, Cytomegalovirus, Herpes simplex virus) were not taken under study. Mycoplasma genitalium, Neisseria gonorrhoeae, Trichomonas vaginalis, Ureaplasma urealyticum, Ureaplasma parvum, Herpes simplex virus ½ were detected in urine by Fast Track Diagnostics STD9 real time PCR kit (Siemens Healthcare, Germany) according to manufacturer's protocol. The amplification was detected in DNA samples by fluorescent reporter dye probes specific to each pathogen with an internal control. Obstetric history information like gravidity, parity, abortion history, and last menstrual period was recorded in questionnaire from all patients enrolled in the study. All experiments were performed in accordance with relevant guidelines and regulations (including informed consent from all participants).
Urine (15-20 ml) was collected from patients enrolled in Groups I and II. Transportation of samples to the laboratory was done on ice. Centrifugation of samples was done at × 1700 rpm for 30 min at 4 °C, the supernatant was discarded and the pellet was stored for further use at − 80 °C.
Chlamydia trachomatis detection in urine. DNA was isolated from urine after dissolving the urine pellet in lysis buffer. Thereafter, DNA was precipitated in propanol and eluted in 50 µl nuclease-free water 23 . Detection of C. trachomatis by conventional PCR was performed in both groups by amplifying chlamydial endogenous cryptic plasmid gene and MOMP gene of 200 and 537 bp respectively. MOMP (5′ TAT ACA AAA ATG GCT CTC TGC TTT AT 3′ and 5′ CCC ATT TGG AAT TCT TTA TTC ACA TC 3′) and plasmid (5′ Table 3. Gene ontology and KEGG analysis of differentially expressed genes associated with SOD2 in recurrent spontaneous abortion. (a) Biological process (b) cellular component (c) molecular function (d) KEGG pathway. www.nature.com/scientificreports/ CTA GGC GTT TGT ACT CCG TCA 3′ and 5′ TCC TCA GGA GTT TAT GCA CT 3′) primer pair sequences were obtained from the published literature 24,25 and these primers were commercially synthesized (Biolink, New Delhi, India). Negative control or no template control and positive control were set up using C. trachomatis DNA (Amplirun Vircell, Spain). Amplification reactions were performed as described earlier 26 .

Detection of Chlamydia trachomatis load in urine by quantitative real-time PCR.
Quantitative real-time PCR (q-PCR) assay was performed to detect the chlamydial load in C. trachomatis-positive urine samples. Serial dilutions (10,000, 1000, 100, 10, 0.1 μl) of C. trachomatis DNA control (Vircell Microbiologist, Granada, Spain) were prepared as per the manufacturer's instructions. 10 μl of SYBR green master mix, 1 μl of plasmid gene (200 bp) forward primer and 1 μl of reverse primer, 5 μl of Vircell C. trachomatis diluted DNA of each concentration and 3 μl of nuclease-free water were mixed to make up a final volume of 20 μl. A standard curve was drawn. By using this standard curve, the chlamydial load was calculated in each sample.
Estimation of 8-OHdG and 8-isolprostane in urine by ELISA. Urine  Quantitative analysis of SOD1 and SOD2 genes by Real-Time PCR. RNA was isolated by Trizol (Invitrogen, USA) method in Group I and II patients according to the manufacturer's guidelines. Briefly, 15-20 ml urine collected was pelleted and thereafter homogenized with 1 ml Trizol and 200 µl chloroform. The supernatant was separated and centrifuged. After addition of 75% ethanol to the supernatant, it was re-centrifuged and the pellet was air-dried. 30 μl of nuclease-free water was used for eluting RNA and the isolated RNA was kept at − 80 °C for future use. Expression of SOD1 and SOD2 genes was studied by real-time PCR assay performed on Step One Plus (Applied Biosystems, USA). A concentration of about 1.5-mg/ml of cDNA was used for performing real time PCR. A 20 μl reaction was set up for the experiment. In brief, 10 μl Sybr Green master-mix, 1 μl of primer, 4 μl of cDNA, and 5 μl of sterile water were added to make up the total volume. Primers sequences (SOD1-5′CGA GCA GAA GGA AAG TAA TG3′ and 5′TAG CAG GAT AAC AGA TGA GT3′; SOD2-5′AGT TCA ATG GTG GTG GTC ATA3′ and 5′CAA TCC CCA GCA GTG GAA TAA3′) were obtained from published literature and commercially synthesized (Biolink, New Delhi, India) 27 . Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) was used as an internal control. The mean threshold cycle (Ct) value was calculated for the target genes and endogenous control and gene expression study was done by relative quantification.
Statistical evaluation. Graph pad Prism (version 8.0) was used for statistical evaluation. Fold change was calculated using 2 − ΔΔCT calculation and analyzed by Mann Whitney test/non-parametric test for comparing mean between two groups. The correlation between SOD genes and GA was analyzed using Spearman's rank correlation test. 'p' value < 0.05 was considered to be significant. PPI with RSA differentially expressed genes (DEGs). To search for potential interacting partners of SOD1 and SOD2 gene, the Search Tool for the Retrieval of Interacting Genes (STRING; http:// string-db. org/) was applied. Functional enrichment analysis by KEGG was studied to decipher the reactome pathways 28 . Four gene expression profiles involved in RSA were retrieved from DEGs using Gene Expression Omnibus (GEO2R). PPI network was generated using the STRING app in Cytoscape. Based on DEGs of four datasets, interaction of SOD1 and SOD2 with DEGs and hub genes was evaluated. After the network was clustered into several groups, Molecular Complex Detection (MCODE; apps.cytoscape.org/apps/mcode) was used to identify the important clusters.

Data availability
The datasets generated used and/ or analysed during the current study available from the corresponding author on reasonable request. All data analysed during this study is included in the article.